JPH01149718A - Liposome preparation easily passing through blood-brain barrier - Google Patents

Abstract

PURPOSE:To provide a liposome preparation capable of easily passing through a blood-brain barrier and prepared by the use of a p-aminophenyl sugar derivative. CONSTITUTION:The objective liposome preparation can be prepared by embedding a beta-galactosidase originated from trumpet shell (Charonia lumpus), jack bean, etc., in a liposome prepared from a p-aminophenyl sugar derivative, e.g., corresponding mannose derivative or galactose derivative. The preparation passes through a blood-brain barrier to supply a brain oligo-dendroglia cell with beta-galactose and is useful as a remedy for Krabbe's disease caused by the deficiency of the galactose.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention utilizes various p-galactosidase embedding carriers.
-Aminophenyl II! The present invention relates to a liposome preparation containing β-galactosidase, which uses liposomes prepared from a derivative, and which easily passes through the blood-brain barrier.

If the preparation of the present invention is administered by intraperitoneal injection, β-galactosidase can be supplied from the blood to brain oligodendroglial cells. Liposomes are mainly composed of phospholipids or certain types of glycolipids, and are formed by suspending a phospholipid solution in water and treating it with ultrasound. It is a membrane or multilayer membrane.

Enzymes such as β-galactosidase (enzymes) are polymeric compounds, so even if they attempt to reach the brain's oligodendroglial cells, their entry is blocked by a barrier called the brain-blood barrier. Therefore, enzymes have traditionally been excluded from the use of therapeutics for brain diseases.

However, the novel form of β-galactosidase-containing liposome preparation according to the present invention completely eliminates the above-mentioned difficulties when administered by intraperitoneal injection. It is extremely useful in clinical therapy as it opens up a new direction in the treatment of Krabbe's disease (in humans).

In the first place, Kratsuba disease is a degenerative brain disease caused by an inborn error of metabolism, and is a disease in which the neutral glycolipid galactocerebroside abnormally accumulates in the oligodendrodarian cells of the central nervous system. , this is a phenomenon caused by a deficiency of β-galactosidase. This defect β
- Supplementation of galactosidase was made possible by administration of the formulation according to the invention.

The liposomes used in the various animal experiments conducted until the completion of the present invention were prepared using lecithin:cholesterol:sulfatide (7:2:1.v) according to the method of Mr. Naoi et al.
/v) or various p-aminophenyl sugar derivatives (mannose, fucose, galactose).

Examples of β-galactosidase to be embedded in these liposomes include Jack bean, Aspergillus oryza, Charonia lumpus, and Bobbin 0 River (Jack
bean, Aspergillus 01izae
Charo-nia Lumpus, Bovine
Liver) et al. The animals used for these experiments include ordinary mice (Hagane mouse) and especially the Twitchel mouse CTwich, which is a model animal for Clatsva disease.
Using a her mouse, the mouse root ganglion cells were cultured according to the method of Kim et al.

Lipid analysis was performed by thin layer chromatography or gas chromatography of glycolipids according to the method of Ledeen et al.

These can be summarized as follows based on the specific results of animal experiments described later.

(a) In Twitchel mice, galactocerebroseeds accumulate in tissues other than the cerebrum.

In human Clatsuba disease, when kidneys were analyzed, galactosereproseed accumulation was found to be 2 to 3 times higher than in controls.

(El) For the treatment of Krappe's disease in mice using liposomes, β-galactosidase derived from the conch shell (Charonia lumpus) was found to be most effective in eliminating excessive accumulation of galactocerebroseeds.

(c) When β-galactosidase derived from conch shell was embedded in sulfatide-containing liposomes, a significant increase in β-galactosidase activity was also observed in liver and lung tissues.

(d) When 3H-galactocerebroside was labeled with liposomes and administered to Twitchel mice, radioactivity in each organ tissue was measured two days later, and radioactivity in the liver and lungs was 40 to 60 times higher than in normal controls. It was found that this could reduce the accumulation of galactocerebroseed.

(f) It has been found that mannose-containing liposomes are more effective than other saccharide-containing liposomes as embedding liposomes for allowing β-galactosidase to efficiently pass through the brain-blood barrier.

Hereinafter, the animal experiments conducted in the present invention will be specifically explained.

(a) In the first experiment, 20×10 cp of 3H-galactocerebroseed-containing liposomes were injected intraperitoneally into each tissue of Twitchel mice and normal mice;
After 2 days, liposome-embedded β-galactosereproseeds were further administered to only half of these mice, and 3H-galactosereproseeds in each organ tissue of these mice were increased over the next 24 and 48 hours. The degree of accumulation and decay of proseed was measured and observed. However, all liposomes used in this experiment were prepared using sulfatide according to the method of Mr. Naoi et al. The results of this experiment are shown in Table 1 below.

As can be seen from this table, the radioactivity of accumulated 3H-galactocerebroseed in the tissues of Twichel mice is 40 to 60 times higher than the corresponding radioactivity in the liver and lungs of normal control mice; Values decreased by 65-85% after 48 hours for mice injected with the enzyme-containing liposomes.

This fact can be understood to indicate that galactosereproseeds accumulated in mouse tissues are destroyed by externally supplying enzyme to mouse tissues. However, it is noteworthy that in this case, no breakdown of galactosereproseed in brain cells occurs, but this is because the liposomes used were prepared by using sulfatide, which could pass through the brain-blood barrier. It can be understood from the results of the second experiment (b) shown below that this is attributable to the fact that

(b) In this second experiment, the liposomes used were prepared using the corresponding mannose derivatives as various p-aminophenyl fPm conductors, and 3H-galactocerebro seeds embedded in these liposomes were injected. After administration, the brain cells and liver cells of the mice were obtained by cell fractionation according to the method of Clendenon et al., and the distribution of radioactivity was measured every 12 hours, 24 hours, 48 hours, and 96 hours. did.

The results are shown in Table 2 below.

2nd Table Brain In this table, the numbers in parentheses indicate the distribution ratio (%) of galactosereproseeds according to radioactivity in each cell fraction. Thus, the radioactivity of the mitochondrial and Rhisosomal fractions is 10-30% in brain tissue and 10-40% in liver tissue, and these peaks are observed within 12-24 hours after injection.

(c) In the third experiment, β-galactosidase derived from conch shell was first administered to normal mice by intraperitoneal injection, and at the elapsed time (immediately, 3 hours, 12 hours, and 24 hours later), each organ of the mouse was The moleproseed β-galactosidase activity was measured. The results are attached in the first part.
Shown in A of the figure.

On the other hand, the β-galactosidase described above was embedded in liposomes prepared using lecithin, cholesterol and sulfatide according to other methods and administered to Mr. Naoi in the same manner as above. The measured Cerebroseed β-galactosidase activity in each organ of the mouse is shown in B of the attached Figure 1.

As can be seen from Figure 1, in case A, the activity in all organs is almost unchanged, but in case B, the activity in the liver, lungs and kidneys is considerably reduced within 24 hours after injection. Increases were observed and they persisted for 3 days (72 hours). However, in the brain, almost no change in activity was observed in either case A or B. This can be understood to indicate that the β-galactosidase is unable to pass through the blood-brain barrier even if it is embedded in a sulfatide-containing liposome and administered.

(d) In the fourth experiment, mannose, fucose (L-form), galactose and fucose (
12 hours, 24 hours, 48 hours and After 72 hours, the radioactivity of (A) brain, (B) liver, (C) kidney, and (D) lung was measured. The results are shown in the attached FIGS. 2 and 3.

As can be seen from (A) in this figure, the activity in brain tissue using mannose-containing liposomes is particularly significantly higher than that in other organs (B), (C), and (D). , and its activity was also recognized in vascular epithelial cells.

It has been found that even high molecular weight enzymes such as β-galactosidase embedded in sugar-containing liposomes can easily pass through the blood-brain barrier. is highly expected to be used as a therapeutic agent for cerebral white matter degeneration, or Clatsuba disease.

[Brief explanation of the drawing]

FIGS. 1, 2, and 3 are line graphs showing temporal changes in enzyme content in mouse organs.

Claims (4)

[Claims] (1) A liposome preparation prepared using a p-aminophenyl sugar derivative that easily passes through the brain-blood barrier. (2) The liposome preparation according to claim (1), wherein the liposome prepared using the p-aminophenyl sugar derivative is a β-galactosidase-containing liposome. (3) Corresponding p- as a p-aminophenyl sugar derivative
The liposome preparation according to claim (1) or (2), prepared using an aminophenylmannose derivative. (4) Conch shell (charo) as β-galactosidase
The liposome preparation according to claim (1), (2) or (3), containing β-galactosidase derived from P. -nialumpus.